Isolation and characterization of bacteriophages against Non O157 Shiga toxin-producing Escherichia coli and their application as biosensor for foodborne pathogen detection

Isolation and characterization of bacteriophages against Non O157 Shiga toxin-producing Escherichia coli and their application as biosensor for foodborne pathogen detection

Author:

Alasiri, Nada

Department:

Department of Food Science

Program:

Food Science

Advisor:

Griffiths, Mansel

Abstract:

The purpose of this study was to determine the potential application of bacteriophages for the detection of Shiga toxin-producing Escherichia coli (STEC) in food. Thirty-five strongly lytic bacteriophages were isolated from various environmental samples against the “big six” STEC serotypes (O111, O121, O103, O145, O26 and O45) and 14 of these phages were chosen for further characterization to determine the most suitable phage for use in a detection assay. The selected phages were characterized for host range, morphology, and adsorption to their bacterial host. One phage (AG2A) was selected from a set of four similar phages as it showed high specificity against its host (E. coli O45:H2). The genome sequence of this phage was determined. The ability of AG2A phage immobilized onto ColorLok paper to detect E. coli O45:H2 in media and food was examined. A phage capture-amplification assay based on the immobilized phage was able to detect as few as 10 CFU/mL of E. coli O45:H2 in both TSB and ground beef using both a plaque assay and real-time PCR to detect phage progeny. The stability of the immobilized phage on the paper was also investigated. It was shown that after one week of storage the detection limit of the assay increased to 50 CFU/mL of E. coli O45:H2 in TSB and ground beef for the PCR-based assay but remained at 10 CFU/mL for the plaque assay. By combining capture of the host using the immobilized phage with detection of progeny phage by real-time PCR, results were obtained within only 8 h. Immobilized phages have great potential for the detection of foodborne bacterial pathogens.